The objective of the proposed research is to develop active-site-directed reagents which are irreversible inhibitors of acyl-CoA utilizing enzymes. The reagents which we synthesize and test are all analogs of CoASH. Highly reactive functional groups, each of which has been shown to react with amino acid side chains by other workers, are incorporated into CoASH, usually at the thiol end of the molecule. Photo-activated functional groups of the aryl-azide type are also attached to CoASH. Formation of an enzyme-reagent complex (covalently bonded) is confirmed by: (a) demonstration of loss of enzyme activity after mixing enzyme and reagent, (b) passing the enzyme and reagent mixture through a sephadex column and analyzing the eluted enzyme for the presence of reagent. The active-site-directed reagents are initially tested as irreversible inhibitors of citrate synthase (pig heart), carnitine acetyltransferase (pigeon muscle), thiolase (beef liver), and short-chain acyl-CoA hydrolase (beef kidney) or long-chain acyl-CoA hydrolase (beef brain). When warranted, the effect of the reagents on other acyl-CoA utilizing enzymes will also be tested. We are particularly interested in developing reagents which will selectively inhibit a certain class of enzyme - for example, a reagent which will inhibit acyl-CoA hydrolases but which will not inhibit acyltransferases. We intend to exploit the active-site-directed reagents for probing the structure and metabolic role of certain acyl-CoA utilizing enzymes.